Insulin receptors and insulin-like growth factor I receptors are functional during organogenesis of the lens

Molecular and Cellular Endocrinology, Elsevier Scientific Publishers Ireland,

MOLCEL

155

14 (1990) 155-162 Ltd.

02403

Insulin receptors and insulin-like growth factor I receptors are functional during organogenesis of the lens Jorge Alemany

I, Matias Girbau

2, Lluis Bassas 2 and Flora de Pablo ’

’ Section on Receptors and Hormone Action, Diabetes Branch, NIDDK,

National Institutes of Health, Bethesda, MD, U.S.A., and ’ Hospital de la Santa Cruz y San Pablo, Barcelona, Spain (Received

Key worak

Insulin;

Insulin-like

growth

factor;

15 May 1990; accepted

Receptor;

11 September

1990)

Lens; Embryo

Summary Insulin and insulin-like growth factor I (IGF-I) stimulate overall growth and development of the chick embryo in early organogenesis. Turning to individual organs, to clarify the cellular effects of these peptides and the activity of the receptors involved, we had demonstrated with developing lens that insulin and IGF-I increase the accumulation of &crystallin mRNA, a marker for lens differentiation, in part by stimulation of transcription. In this study we expand our previous work on lens receptors to an earlier time in organogenesis, day 4, which marks the beginning of differentiation of the lens epithelial cells into elongated fibers. Insulin receptors are demonstrable by affinity cross-linking in epithelial cells at day 6, and specific binding of [‘251]insulin and [‘251]IGF-I is detectable in day 4 lenses. Insulin and IGF-I stimulation of substrate phosphorylation in the presence of solubilized receptors occurs only with high concentrations (lo-100 nM) of either peptide in day 4 lenses, while a clear response with low concentrations (1 nM) is elicited by day 6 of development. Low concentrations of both insulin and IGF-I (0.1-l nM) increase the incorporation of [3H]leucine and [3H]uridine in day 6 lens cells, suggesting that each peptide acts through its own receptor. These results confirm and extend the finding of insulin and IGF-I receptors in the developing chicken lens, and demonstrate their functional activity. This embryonic model should be valuable for further analysis of the action of insulin and IGF-I in growth and differentiation processes during early development.

Introduction The insulin receptor and the insulin-like growth factor I (IGF-I) receptor are members of a superfamily of membrane-associated tyrosine kinases

Address for correspondence: Flora de Pablo, Bldg. 10, Room 88-243, National Institutes of Health, Bethesda, MD 20892, U.S.A.

0303-7207/90/$03.50

0 1990 Elsevier Scientific

Publishers

Ireland,

involved in the control of normal and abnormal cell growth, that includes several hormone and growth factor receptors and proto-oncogenes (Yarden and Ullrich, 1988; Ullrich and Schlessinger, 1990). The mature receptors for insulin and IGF-I have similar heterotetrameric structures with two extracellular a-subunits, containing the ligand binding domain, and two P-subunits, which span the plasma membrane and contain the cytoplasmic tyrosine kinase domain (Massague and Ltd.

156

Czech, 1982). These tyrosine kinases are capable of autophosphorylating the receptor’s P-subunit and phosphorylating many endogenous and exogenous substrates (Zick et al., 1985; Kasuga et al., 1990). Although each receptor has a high affinity for its homologous ligand (and lower affinity for the heterologous), both receptors bind insulin and IGF-I. Some intracellular substrates are also common to both types of receptors (Kadowaki et al., 1987). These characteristics lead to an overlapping spectrum of actions mediated by the two receptors, ranging from metabolic effects to mitogenesis and differentiation (Pepe et al., 1987; Corps and Brown, 1988; Kahn and Harrison, 1988; Lammers et al., 1989; Thies et al., 1989). There may be, however, intrinsic differences in the signal transduction capability of the insulin receptor and the IGF-I receptor (Lammers et al., 1989). A normal tyrosine kinase activity is considered essential for the transduction of the hormonal signal leading to many intracellular effects (Morgan et al., 1986; Chou et al., 1987). During chicken embryo development, both insulin receptors and IGF-I receptors are found widely distributed (Bassas et al., 1988; Girbau et al., 1989). Further, insulin (De Pablo et al., 1982) insulin mRNA (Serrano et al., 1989), IGF-I and IGF-I mRNA (Serrano et al., 1990) are also present in the embryo during early organogenesis. Thus, it is likely that insulin and IGF-I play a coordinate role in the control of cell growth and differentiation of a variety of tissues. To analyze this role, the lens of the eye has some unique and advantageous characteristics. The lens develops very early in vertebrates as an isolated organ, lacking blood vessels and nerves (Johnston et al., 1979). In the chicken, at day 3 of embryogenesis the lens detaches from the underlying ectoderm and forms a vesicle of epithelial cuboidal cells which progressively differentiate into elongated fiber cells forming the bulk of the lens by day 6 of embryogenesis (Fig. 1). While the epithelial cells are mitotically active, the fully differentiated fibers are amitotic and synthesize large amounts of the specialized protein S-crystallin (Nath et al., 1987; Wistow and Piatigorsky, 1988). We have previously demonstrated specific binding of insulin and IGF-I to crude membrane preparations from epithelial and fiber lens cells (Bassas

A

B

F

Fig. 1. Chicken lens organogenesis. A: Diagram of the lens and surrounding structures in a chicken embryo at 3-4 days of development. The eye forms an independent structure with clearly distinguishable components. The virtual space between the non-differentiated epithelial cells and the newly differentiated fibers, closes at day 4 approximately. L, lens; VH, vitreous humor; PR, pigmented retina; NR, neural retina. (Adapted from Piatigorsky, 1981.) B: Microphotography of a sagittal section (6 pm) of a 6-day-old chicken embryo lens. The cuboidal non-differentiated epithelial cells are localized in the centralfront part of the lens. As they differentiate, they elongate, lose their cytoplasmic organelles and accumulate large amounts of crystallins, conforming the bulk of the lens. At this early age, the fiber cells still maintain the nucleus, which is lost in later stages. E, epithelial cells; F, fibers; C, cornea. Magnification x 125.

1.57

et al., 1987). The binding of the two ligands changed with developmental age, and also at each age, depending on the stage of cell differentiation, i.e., IGF-I binding was highest at the earliest age studied, day 6 of embryogenesis and decreased in older embryos in both epithelial and fiber cells. Insulin binding did not change markedly with age but it was much lower at each age in differentiated fibers epithelial cells. More recently, we have provided some evidence that the effects of insulin on the expression of the &crystallin gene in embryonic lens are receptor-mediated (Alemany et al., 1989, 1990). Other eye structures such as the retina (Kyriakis et al., 1987; Bassas et al., 1989) and sclera (Waldbillig et al., 1990) display insulin receptors and IGF-I receptors, both of which appear to dominate also in tissues of younger embryos. In the present study we characterize further the insulin receptors and IGF-I receptors of the developing lens. We show that insulin- and IGF-I-dependent tyrosine kinase activities are well established by day 6 of embryogenesis in the lens. The incorporation of .[3H]uridine and [3H]leucine in lens cells is stimulated by both insulin and IGF-I, at low concentrations.

Affinity crosslinking of [“‘I]insulin

to receptors

Aliquots of membranes from day 6 embryo lens epithelium containing 800 pg,/ml of protein were incubated with 3.2 ng,/ml ~12sI]insulin and the ligand-receptor complex was crosslinked with 0.1 mM disuccinimidyl suberate, pH 8, as described (Bassas et al., 1987). Aliquots of membranes from embryonic brain and liver were processed in parallel. Samples were solubilized by boiling at 100 o C for 6 min in a solution of 125 mM Tris, 10% glycerol and 2% sodium dodecyl sulfate (SDS) in the presence of 100 mM dithiothreitol. Samples were electrophoresed in one-dimensional, discontinuous SDS-polyac~la~de slab gel according to Laemmli (1970). The gel exposed film to generate

800 from day were recycled over with the

from and

whole lenses from day fractions were pooled and

were incubated for min at room temperature in 50 mM Hepes buffer (pH 7.6) the presence

Materials and methods

with or without the adPreparation of membranes and solubilized receptors from lens cells

White leghorn chicken eggs were obtained from Truslow Farms (Chesterton, MD, U.S.A.). Lenses from 4- and 6-day-old embryos (hatching is at day 21) were removed from the eye by microdissection. For the crosslinking study the anterior epithelium was ~crodissected and crude membranes were prepared as described (Bassas et al., 1987). For the phosphorylation studies whole lenses were homogenized and solubilized receptors were partially purified by chromatography over wheat germ agglutinin (WGA) as described (Girbau et al., 1989). Binding of [‘251]insulin and [lz51]IGF-I

Aliquots of WGA eluates were used for binding experiments with 0.04-0.05 nM [1251]insulin or [‘2SI]IGF-I, as described (Girbau et al., 1989).

dition 140 ~1 following established al., 1985; Girbau al., 1989). Stimulated tyrosine the difference between the the presence and the stimulating peptides, insulin 1000 nM or nM ~ncentration respectively. [.‘Ii]Leucine

were microdissected from day and cultured the absence F-10 defined medium at 37 ’ C, 5% 24 h. medium was replaced (80 pCi/ml) and the cells were cubated for one additional hour the same conditions. The was

158

measured after the cells were washed trichloroacetic acid and homogenized.

with

5%

[-‘HI Uridine uptake Epithelial and fiber cells from five lenses were microdissected as previously described ,(Piatigorsky, 1981). The cells were incubated for 2 h at 37” C, 5% CO,, in 2 ml Ham’s F-10 medium supplemented with 80 pCi/ml [‘Hluridine (Amersham) in the presence or absence of a range of concentrations of insulin and IGF-I. After incubation the cells were washed twice with phosphate buffered saline (PBS), pH 7.4, centrifugated and lysed with 250 ~1 of 1 N NaOH (at 37 o C for 15 mm). Duplicate aliquots were taken from the lysate, the radioactivity was measured in a scintillation counter and it was expressed relative to protein content. Results

Insulin and IGF-I binding to lens cells Binding of [‘251]insulin and [1251]IGF-I to receptors from lenses of day 4 and day 6 embryos (Table 1) confirmed and extended our previous studies in crude membranes (Bassas et al., 1987). With solubilized WGA-selected receptors, the binding of [‘251]IGF-I is multifold higher than [i2’I]insulin binding in whole lens. Lenses from day 4 embryos showed similar binding to day 6 lenses for the two ligands. Affinity crosslinking of [ 125I]insulin demonstrated in epithelial cells an a-subunit of the insulin receptor with an apparent between the liver M, of - 129,000, intermediate receptor and the brain receptor, presumably due

TABLE

Br

Li

to differences in the carbohydrate receptor-glycoprotein (Fig. 2).

1

SPECIFIC BINDING OF LABELLED PEPTIDES WHOLE LENS SOLUBILIZED RECEPTORS

TO

Aliquots of WGA eluates were incubated with labelled peptides in the absence (total binding) or in the presence (nonspecific binding) of an excess of unlabelled homologous ligand. The nonspecific binding (range 7-248 of the total binding) was subtracted to obtain the specific binding. The mean value of duplicate points is shown.

Day 4 Day 6

E

Fig. 2. Affinity labelling of insulin receptors. [‘251]Insulin was crosslinked to membrane preparations from lens central epithelium, 6-day-old embryo brain and 6-day-old embryo liver. The autoradiogram of the polyacrylamide gel (6.5%) exposed to X-ray film is shown. The arrow indicates the a-subunit of insulin receptors in lens epithelium (E), brain (Br) and liver (Li). The position of the molecular weight standards is indicated.

6.6% 4.7%

56.1% 52.2%

content

of the

Ligand-stimulated phosphorylation of exogenous substrate (poly(Glu, Tyr)) by solubilized receptors from developing lens We analyzed the ability of the lens receptors to transduce the hormonal signal by studying an early postbinding step, i.e. their tyrosine kinase activity. The incorporation of 32P into the artificial substrate poly(Glu,Tyr) was stimulated in a dose-dependent fashion by insulin and IGF-I in the presence of WGA-purified receptors. Insulin and IGF-I had similar potency, with a tendency of

159

IGF-I to have a higher effect, more evident 6 (Fig. 3).

at day

r3H]Uridine uptake and[3H]leucine uptake are increased by insulin and IGF-I Insulin and IGF-I, at low nanomolar concentrations, stimulated by 2-fold [ ‘Hlleucine uptake, an indirect measure of protein synthesis, into day 6 lens epithelial cells (Fig. 4). The [3H]uridine uptake, an indirect measure of RNA synthesis,

Day 4 Embryo Lens

--_ll;I 1

10

100

1000

1

a, H

10

100

IGF-I (nM)

INSULIN (nM)

200

Dav 6 Embrvo

1

10

100

INSULIN

1000

(nM)

1

10

100

IGF-I (nM)

Fig. 3. Insulin and IGF-I stimulation of exogenous substrate phosphorylation. WGA-purified receptor preparations from lenses of 4- and 6-day-old embryos were incubated in the presence of insulin or IGF-I at the concentrations indicated. The figure shows the fmol of ATP incorporated to the exogenous substrate. In each case the phosphorylation of endogenous substrates in the preparations was determined, and this value was subtracted to obtain the estimulated incorporation of 32P into exogenous substrate. The results for day 6 lens are the mean of four separate experiments done with different lens preparations, each consisting of 400 lenses. The results for day 4 lens are the mean of duplicate determinations in a single experiment in which 800 lenses were used. (Note that the concentrations of peptides are presented in this figure in nM; for insulin, 1 nM = 6 ng/ml, and for IGF-I, 1 nM = 7.5 ng/mU

CONTROL

-/1 10 INSULIN kg/ml)

1

10

IGF-I Mk7imll

Fig. 4. Insulin and IGF-I effect on [‘Hlleucine uptake. Microdissected epithelial cells from 6-day-old chick embryo lenses were incubated for 24 h in the presence or absence of insulin or IGF-I at the indicated concentrations. After one additional hour of incubation in the presence of [3H]leucine, the cells were washed, homogenized and the radioactivity associated with the cells was measured in a scintillation counter. Values are referred to protein content; the mean value of four experiments and the standard error are indicated.

was increased about 2- to 3-fold in the presence of both hormones (Fig. 5). The [3H]uridine uptake per pg of protein is higher in epithelial cells, both in the basal and the stimulated states, reflecting the higher general cellular activity of these cells. Although not statistically significant, IGF-I showed a slightly higher effect than insulin. In a single pilot experiment, the [ 3H]uridine uptake into day 4 embryo lenses was not modified by the presence of IGF-I. Discussion We report the presence of functional receptors and IGF-I receptors in chicken lens during organogenesis. At this stage differentiation, both types of receptors ligands, contain active tyrosine kinases, pear to mediate insulin and IGF-I effects lar activities. While the lens is structurally a simple has a complex developmental regulation,

insulin embryo of tissue bind the and apon celluorgan it in which

160

I

0

Eplthellal

0.1

Cells

1.0

/

Fiber Cells

10

PEPTIDE CONCENTRATION

ing/mli

Fig. 5. Insulin and IGF-I effects on [3H]uridine uptake. Microdissected epithelial and fiber cells from 6-day-old chick embryo lenses were incubated for 2 h with 80 &i/ml of [ ‘Hluridine in Ham’s F-10 medium with the addition of a range of concentrations of insulin or IGF-I. After precipitation with 5% trichloroacetic acid, the radioactivity incorporated into the cells was measured. The main figure shows the incorporation in treated cells relative to untreated cells, after the values in all groups were normalized per protein content. In the inset. the effects of 10 r&ml of insulin and 10 ng/ml IGF-I are represented as actual cpm relative to protein, to show the absolute levels of maximal stimulation. The mean of values obtained in three experiments and the standard error are indicated.

several hormones and growth factors are involved. Epidermal growth factor (EGF) and platelet derived growth factor (PDGF) participate in the control of growth ,and postnatal development of the lens (Hollenberg, 1975; Reddan and WilsonDziedzic, 1983; Brewitt and Clark, 1988). Initial studies on the process of lens differentiation during embryogenesis in chicken showed that fetal calf serum, vitreous humor extract and insulin, all induced the epithelial cells to elongate and become specialized fibers that synthesize large amounts of b-crystallin (Milstone and Piatigorsky, 1979; Beebe and Feagans, 1981). Later, a vitreous humor component, named lentropin, with the immunological characteristics of IGF-I, as well as recombinant human IGF-I, were shown to be potent stimulators of lens morphological differentiation (Beebe et al., 1987). Recently, we studied the ability of insulin and IGF-I to regulate 6crystallin gene expression, a marker of differentiation in lens cells. Both hormones increased the accumulation of S-crystallin mRNA to a similar extent (Alemany et al., 1989) but IGF-I was more

effective activating transcription of the 61-crystallin gene (Alemany et al., 1990). Due to the difficulty in obtaining large quantities of tissue from young embryo lens, our initial study on the receptors for insulin and IGF-I was limited to the developmental profile of the ligands binding to crude membrane preparations (Bassas et al., 1987). In the present study, we have further analyzed the function of the lens receptors in mid-organogenesis (day 6) and we have initial information on the youngest lens, day 4, at the beginning of organogenesis. Despite similar level of specific binding of the two ligands at day 4, compared to day 6, the response to low concentrations of the hormones in the phosphorylation assay was not clearly established until day 6 of embryogenesis. In addition, the specific binding of insulin is much lower than the specific binding of IGF-I, while the stimulations of the kinases are closer. (Note that the binding of insulin is IO-fold less than the binding of IGF-I while in our previous study (Bassas et al., 1987) done with crude membranes, there was only a 2- to 3-fold difference. It is possible that not all insulin receptors bind to WGA and, therefore, we have preferentially enriched our preparation for IGF-I receptors.) These data suggest that there are developmental changes in the coupling of the CV- and P-subunits, and that there are differences between insulin receptors and IGF-I receptors in that process. We had previously shown that the whole day 4 embryo contains insulin and IGF-I receptors with stimulatable tyrosine kinases. The activities elicited by insulin and IGF-I were multifold higher than in younger (day 2) whole embryos (Girbau et al., 1989). Therefore, there is developmental regulation of the acquisition of tyrosine kinase activity of the insulin receptor and IGF-I receptor that may be tissue-specific. Other organs more developed than the lens at day 4, the heart for example, may be main contributors to the overall embryo ligand-stimulated tyrosine kinase activity. Interestingly, in Drosophila whole embryos there is also early expression of the insulin receptor (Petruzzelli et al., 1985) which exhibits developmentally regulated kinase activity, sensitive to insulin. We do not address directly in our present studies how the tyrosine kinase activities relate to the

161

biological action of the peptides. In other systems it remains a controversial issue. Insulin receptors with defective tyrosine kinase or mutated in the ATP binding site do not mediate some biological effects well (Maegawa et al., 1988; McClain et al., 1988). In contrast, some mutations in the P-subunit of the receptor, which do not affect the autophospho~lation or the phosphotransferase activities, have impaired metabolic action (White et al., 1988; Thies et al., 1989). Our results indicate that the potency of insulin in the kinase assay was a better predictor of the cellular effects of the hormone than the [‘251]insulin binding level, i.e. the ability to stimulate [ 3H]leucine and [ 3H]uridine inco~oration was very similar for insulin and IGF-I in day 6 lens cells. Interestingly, in chick embryo hepatocytes, others have found stimulation of [14C]valine and [~Hluridine inco~oration to the same extent by treatment with insulin and IGF-I (Widiner et al., 1985). The low concentration effective in our system suggests that each peptide is acting through its own receptor. The importance of insulin and IGF-I effects in early development is starting to be recognized. Insulin at low concentrations has recently been found to stimulate inco~oration of macromolecules in preimplantation mouse embryos (Harvey and Kaye, 1988; Heyner et al., 1989). In the lens system, further studies in vivo such as interference of lens differentiation by anti-IGF-I and anti-insulin antibodies may provide clues to the specific requirements of each hormone in normal lens organogenesis. Acknowledgements

We thank Maxine A. Lesniak for comments on the manuscript and Jesse Roth for generous support to this project. These studies have been partially funded by the U.S.-Spain Joint Committee for Scientific and Technolo~cal Cooperation. References Alemany, J., Zelenka, P., Serrano, J. and De Pablo, F. (1989) .I. Biol. Chem. 264, 17559917563. Alemany, J.. Borras, T. and De Pablo, F. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3353-3357.

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